Enveloped viruses depend upon fusion between the viral membrane and a host cell membrane (the plasma membrane or an intracellular membrane, depending upon the specific virus) for delivery of viral genetic material to the host cell, thereby initiating infection of the host cell (Kielian & Ray, “Virus Membrane-fusion Proteins: More Than One Way to Make a Hairpin,” Nat. Rev. 4:67-76 (2006), which is hereby incorporated by reference in its entirety). This membrane-fusion reaction relies on virus membrane-fusion proteins (Kielian & Ray, “Virus Membrane-fusion Proteins: More Than One Way to Make a Hairpin,” Nat. Rev. 4:67-76 (2006), which is hereby incorporated by reference in its entirety). At least two classes of membrane-fusion proteins have been identified (Kielian & Ray, “Virus Membrane-fusion Proteins: More Than One Way to Make a Hairpin,” Nat. Rev. 4:67-76 (2006), which is hereby incorporated by reference in its entirety). Class I fusion proteins contain two heptad repeat regions, termed the N-terminal heptad region and the C-terminal heptad region, between a hydrophobic “fusion peptide” region and a transmembrane domain (Dutch et al., “Virus Membrane Fusion Proteins: Biological Machines That Undergo a Metamorphosis,” Biosci. Rep. 20(6):597-612 (2000), which is hereby incorporated by reference in its entirety). During membrane fusion, these heptad repeat regions ultimately adopt a highly stable coiled-coil assembly in which the N-terminal heptad repeat region forms an internal, trimeric coiled-coil buttressed by helices from the C-terminal heptad repeat region (Dutch et al., “Virus Membrane Fusion Proteins: Biological Machines That Undergo a Metamorphosis,” Biosci. Rep. 20(6):597-612 (2000), which is hereby incorporated by reference in its entirety). Agents that interfere with the formation of this coiled-coil assembly can prevent viral-host cell membrane fusion, thereby inhibiting infection of the new host cell.
Viruses that use class I coiled-coil assemblies for viral infectivity include: Orthomyxoviridae, e.g., influenza virus (Dutch et al., “Virus Membrane Fusion Proteins: Biological Machines That Undergo a Metamorphosis,” Biosci. Rep. 20(6):597-612 (2000); Kielian & Ray, “Virus Membrane-fusion Proteins: More Than One Way to Make a Hairpin,” Nat. Rev. 4:67-76 (2006), which are hereby incorporated by reference in their entirety); Paramyxoviridae, e.g., Simian virus 5 (Dutch et al., “Virus Membrane Fusion Proteins: Biological Machines That Undergo a Metamorphosis,” Biosci. Rep. 20(6):597-612 (2000); Kielian & Ray, “Virus Membrane-fusion Proteins: More Than One Way to Make a Hairpin,” Nat. Rev. 4:67-76 (2006), which are hereby incorporated by reference in their entirety) and respiratory syncitial virus (Dutch et al., “Virus Membrane Fusion Proteins: Biological Machines That Undergo a Metamorphosis,” Biosci. Rep. 20(6):597-612 (2000); Shepherd et al., “Modular α-Helical Mimetics with Antiviral Activity Against Respiratory Syncitial Virus,” J. Am. Chem. Soc. 128:13284-9 (2006), which are hereby incorporated by reference in their entirety); Filoviridae, e.g., Ebola virus (Dutch et al., “Virus Membrane Fusion Proteins: Biological Machines That Undergo a Metamorphosis,” Biosci. Rep. 20(6):597-612 (2000); Kielian & Ray, “Virus Membrane-fusion Proteins: More Than One Way to Make a Hairpin,” Nat. Rev. 4:67-76 (2006), which are hereby incorporated by reference in their entirety); Retroviridae, e.g., Moloney murine leukemia virus, simian immunodeficiency virus, Human immunodeficiency virus (“HIV-1”), and human T cell leukemia virus (Dutch et al., “Virus Membrane Fusion Proteins: Biological Machines That Undergo a Metamorphosis,” Biosci. Rep. 20(6):597-612 (2000); Kielian & Ray, “Virus Membrane-fusion Proteins: More Than One Way to Make a Hairpin,” Nat. Rev. 4:67-76 (2006), which are hereby incorporated by reference in their entirety); Coronaviridae, e.g., Mouse hepatitis virus and SARS virus (Kielian & Ray, “Virus Membrane-fusion Proteins: More Than One Way to Make a Hairpin,” Nat. Rev. 4:67-76 (2006), which is hereby incorporated by reference in its entirety); and Herpesviridae, e.g., human cytomegalovirus (English et al., “Rational Development of β-Peptide Inhibitors of Human Cytomegalovirus Entry,” J. Biol. Chem. 281:2661-7 (2006), which is hereby incorporated by reference in its entirety).
HIV-1 is illustrative of viruses that use class I fusion proteins. HIV has been identified as the etiological agent responsible for acquired immune deficiency syndrome (“AIDS”), a fatal disease characterized by destruction of the immune system and the inability to fight off life threatening opportunistic infections. Recent statistics indicate that as many as 33 million people worldwide are infected with the virus (AIDS EPIDEMIC UPDATE at 1, United Nations Programme on HIV/AIDS (December 2007)). In addition to the large number of individuals already infected, the virus continues to spread. Estimates from 2007 point to close to 2.5 million new infections in that year alone (AIDS EPIDEMIC UPDATE at 1, United Nations Programme on HIV/AIDS (December 2007)). In the same year there were approximately 2.1 million deaths associated with HIV and AIDS (AIDS EPIDEMIC UPDATE at 1, United Nations Programme on HIV/AIDS (December 2007)).
Entry of HIV-1 into its target cells to establish an infection is mediated by viral envelope glycoprotein (“Env”) and cell surface receptors (CD4 and a coreceptor, such as CXCR4 or CCR5) (Eckert & Kim, “Mechanisms of Viral Membrane Fusion and Its Inhibition,” Annu. Rev. Biochem. 70:777-810 (2001), which is hereby incorporated by reference in its entirety). The mature Env complex is a trimer, with three gp120 glycoproteins associated non-covalently with three viral membrane-anchored gp41 subunits. Binding of gp120/gp41 to cellular receptors triggers a series of conformational changes in gp41 that ultimately leads to formation of a postfusion trimer-of-hairpins structure and membrane fusion (Chan et al., “Core Structure of gp41 from the HIV Envelope Glycoprotein,” Cell 89:263-73 (1997); Weissenhorn ct al., “Atomic Structure of the Ectodomain from HIV-1 gp41,” Nature 387:426-30 (1997); Tan et al., “Atomic Structure of a Thermostable Subdomain of HIV-1 gp41,” Proc. Nat'l Acad. Sci. U.S.A. 94:12303-8 (1997), which are hereby incorporated by reference in their entirety). As shown in FIGS. 1A and 1C, the core of the postfusion trimer-of-hairpins structure is a bundle of six α-helices: three N-peptide helices form an interior, parallel coiled-coil trimer, while three C-peptide helices pack in an antiparallel manner into hydrophobic grooves on the coiled-coil surface (Chan et al., “Core Structure of gp41 from the HIV Envelope Glycoprotein,” Cell 89:263-73 (1997); Weissenhorn et al., “Atomic Structure of the Ectodomain from HIV-1 gp41,” Nature 387:426-30 (1997); Tan et al., “Atomic Structure of a Thermostable Subdomain of HIV-1 gp41,” Proc. Nat'l Acad. Sci. U.S.A. 94:12303-8 (1997), which are hereby incorporated by reference in their entirety). The N-peptide region features a hydrophobic pocket targeted by C-peptide residues W628, W631, and 1635, as shown in FIG. 1B (Chan et al., “Evidence That a Prominent Cavity in the Coiled Coil of HIV Type 1 gp41 Is an Attractive Drug Target,” Proc. Nat'l Acad. Sci. U.S.A. 95:15613-7 (1998), which is hereby incorporated by reference in its entirety). Agents that interfere with the formation of the gp41 coiled-coil hexamer are primary targets for vaccine and drug development (Deng et al., “Protein Design of a Bacterially Expressed HIV-1 gp41 Fusion Inhibitor,”Biochem. 46:4360-9 (2007), which is hereby incorporated by reference in its entirety). Peptides and synthetic molecules that bind to the N-terminal hydrophobic pocket and inhibit the formation of the six-helix bundle have been shown to effectively inhibit gp41-mediated HIV fusion (Wild et al., “Peptides Corresponding to a Predictive α-Helical Domain of Human Immunodeficiency Virus Type 1 gp41 Are Potent Inhibitors of Virus Infection,” Proc. Nat'l Acad. Sci. U.S.A. 91:9770-4 (1994); Ferrer et al., “Selection of gp41-mediated HIV-1 Cell Entry Inhibitors from Biased Combinatorial Libraries of Non-natural Binding Elements,” Nat. Struct. Biol. 6:953-60 (1999); Sia et al., “Short Constrained Peptides That Inhibit HIV-1 Entry,” Proc. Nat'l Acad. Sci. U.S.A. 99:14664-9 (2002); Ernst et al., “Design of a Protein Surface Antagonist Based on α-Helix Mimicry: Inhibition of gp41 Assembly and Viral Fusion,” Angew. Chem. Int'l Ed. Engl. 41:278-81 (2002), originally published at Angew. Chem. 114:282-91 (2002); Frey et al., “Small Molecules That Bind the Inner Core of gp41 and Inhibit HIV Envelope-mediated Fusion,” Proc. Nat'l Acad. Sci. U.S.A. 103:13938-43 (2006); Stephens et al., “Inhibiting HIV Fusion with a β-Peptide Foldamer,” J. Am. Chem. Soc. 127:13126-7 (2005); Deng et al., “Protein Design of a Bacterially Expressed HIV-1 gp41 Fusion Inhibitor,” Biochem. 46:4360-9 (2007), which are hereby incorporated by reference in their entirety).
However, not all patients are responsive to existing therapies, and the virus develops resistance to most, if not all, known agents. Thus, there is a need for new antiviral agents against HIV and other viruses that use class I fusion proteins.